manual - DNA Technologies Core
Contents
1. Version 1 5 Bioruptor NGS Cat No UCD 600 Guarantee Limited one year global warranty Diagenode guarantees all products from any manufacturing defects as we rigorously test all products to meet strict quality standards Diagenode warrants that all standard components of its instruments will be free of defects in materials and workmanship for a period of one 1 year from the date that the warranty period begins unless the original quotation or accompanying documentation states a different warranty period All warranty periods begin on the date of delivery and apply only to the first purchaser of the product If a manufacturing defect arises and a valid claim is received within the warranty period Diagenode at Its discretion will repair or replace the product in accordance with the warranty terms and conditions stated herein In case of repair or replacement of a product under warranty Diagenode will cover the expenses to return the repaired or replacement product This warranty covers only manufacturing defects and does not cover any damage caused by misuse lack of compliance to recommendations stated in the manual neglect accidents abrasion or exposure to extreme temperatures chemical solvents or acids We strongly recommend that maintenance or repairs of Diagenode s products are performed by our approved Diagenode service center Improper or incorrectly performed maintenance or repairs will void the warranty Technical Assist2. or GC rich regions 2 The analytical process to determine fragment size has inherent variances for example gel electrophoresis and microfluidics based platform Therefore fragment distributions and peak values even from technical replicates may not appear identical If the sheared DNA sample will be resin or column purified or concentrated prior to analysis please remember to take out an aliquot for use as control prior to that step Column purification and concentration of the sheared DNA will generate a biased fragment distribution profile due to the Inherent greater loss of the smaller DNA fragments 3 RNA contamination in genomic DNA preparation should be carefully removed using RNase DNase free enzymatic digestion since they might generate a biased fragment distribution profile on microfluidics based platform e g Agilent Bioanalyzer or alter sonication effiency Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR NGS USER MANUAL PAGE 19 Related Products Diagenode develops and sells premium products for Epigenetics research that provide industry leading sensitivity and consistency Diagenode offers a number of kits for Chromatin Immunoprecipitation ChIP and DNA methylation assays like Methylated DNA immunoprecipitation MeDIP MethylCap MBD and Bisulfite conversion MagBisulf
3. ao a MMM MIZ MMMMWNN ITJHDDNM 0 9 Equipment iInstablatl N osorno roces aa a E 10 Installation Overvlew kk kk kk kk kk KK KK KK KK KK KK KK kK kk kk kk kk kk kk kk kk kK kK kK kK KK KK KK KK k 10 Installing the Bioruptor NGS System with Water Cooler WW kk kk kK KK KK KK KK 11 Controlling ING SONICANONs e nuda ode sk ads io a kur oo bs ots dar sra onan se id d jn dd 14 TUDE Holders and T y ys css ke sa ks n kah dal a ln aa di xd db tad ado aa 15 Standard protocol for DNA Shearing kk mk kk kk kk kk kk kK kK KK kk kk kk kk kk kk kk kk kk e 15 Opera tna Conditi e oo k lan kak Xana Z nn bak h dena y k hb n ide Ra kh K ara Wore a etd 15 Important comments about DNA shearing 0 0 200 eee 16 Bioruptor Packing lnstruct ons sois atadas kk kk anann kk Ra kk a kk kk kk kk kk kk kk kk a 18 Related Products AMEN kk kk kk kk kk kK kK kK kK kK KK KK kk kk kk lk kk kk kk lk lk lk kk ok kk lk kk kk kk lk lk kk lk ka 19 DNA Mel l CHO Tl N LS lt ac ak su rana 22A u haaa kard wl a Eee ll k dilara k KWE ana Qu hk Mann ed dere aa wl d Were di dw hd di 19 O 19 Ordering Inf rmalo so rs aa A at ia Back Cover Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 4 DIAGENODE BIORUPTOR NGS USER MANUAL Critical Steps for Maintenance and Efficient Shearing General warnings AN FRAGILE e DO NOT turn on the instrument without water
4. as microarray and sequencing 2 Hydroxymethylated methylated and unmethylated BAC clones Hydroxymethylated DNA control methylated DNA control unmethylated DNA control Sfi1 for genomic DNA 16 AF Auto02 0016 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 22 DIAGENODE BIORUPTOR NGS USER MANUAL Manual DNA methylation kits Features Suitable for Amount of DNA rxn Total Time of Assay Handling time Internal controls Control PCR primer pairs rxns per kit Cat No control primer pairs analysis microarray Depending on the DNA purification method MethylCap kit MagMeDIP kit Allows to capture fractions of methylated DNA by CpG density magnetic beads permit Quality control using internal controls improved handling magnetic beads high specificity monoclonal 5 meC Ab fast fast and sensitive capture includes for assessment of capture efficiency qPCR linear amplification and genome wide qPCR linear amplification and genome wide analysis microarray sequencing Depending on the DNA purification method sequencing selected selected lg 1 pg 1 day 2 or 3 days 2h Lon Methylated and unmethylated BAC clones Methylated DNA control unmethylated TSH2B GAPDH DNA control TSH2B GAPDH 48 10 or 48 AF 100 0048 ak cal mec magme 048 MeDIP kit Quality control using internal controls h
5. bath Output selector switch Power plug must be grounded POLLUTION DEGREE 2 Normally only non conductive pollution occurs However occasionally a temporary conductivity caused by condensation is expected Never install this equipment in a place where environmental conditions and warnings mentioned above are infringed Connector kit for Bioruptor Water Cooler Bioruptor Water Cooler Power cord Fig 4 Schematic installation overview of the Bioruptor NGS System in combination with the Bioruptor Water Cooler and the Connector kit Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR NGS USER MANUAL PAGE 11 Installing the Bioruptor NGS System with Water Cooler 1 Open the boxes and unpack all components 2 Place water bath in front 3 Remove the rubber cap upper left hole marked machine cable from the of the soundproof box back of the soundproof box and feed the control unit cable through the hole 4 Plug the control unit cable into the water bath Make sure the red arrow on the plug is pointing up wa 5 Place the 2 black plastic connectors on the motorized lid Both connectors can be placed into one or the other hole of the motorized lid Screw the plastic nut over the motorized lid The short red and blue pieces of tubing have to be I
6. chip Sample volume 100 ul Tubes Bioruptor NGS 0 65 ml Microtubes for DNA Shearing Cat No WA 005 0500 Tube holder 0 5 0 65 ml tube holder Cat No UCD pack 0 5 for 12 x 0 65 ml tubes Sonication buffer TE 10 mM Tris 1mM EDTA pH 7 5 8 0 DNA concentration 0 001 0 02 ug ul 0 01 ug ul recommended Samples are vortexed 10 15 sec and centrifuged 10 sec before shearing For optimal results samples should be stored on ice during 10 15 minutes prior to sonication Temperature 4 C Bioruptor Water Cooler Cat No BioAcc Cool amp Single cycle valve for Water Cooler Cat No VB 100 0001 Power setting H position High Sonication cycle amp total sonication time varies depending on desired DNA size see table Note Recommended protocols are subject to change without notice Additional protocols are avallable on demand Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR NGS USER MANUAL PAGE 17 7 Cycle condition 150 bp 30 30 30 200 bp 30 30 13 400 bp 157 90 7 8 1000 bp 5 90 E For longer fragments 400 up to 1000 bp a short centrifugation step after half of the cycle numbers can significantly improve the results Protocols for other size ranges incl longer fragments up to 1300 bp are available on request The protocol settings listed above
7. e DO NOT tilt the water bath To change the water use either the plastic pump or a beaker Water bath levels e The water bath must be filled with distilled water to the fill line Fill line replacement stickers can be obtained by contacting Diagenode e Change water at least once per week Water bath temperature e Optimal temperature for sonication is 4 C Sample temperature should not exceed 10 C e The Diagenode Water Cooler Cat No BioAcc Cool has to be used in combination with the Connector kit for Water Cooler Bioruptor Plus NGS Cat No VB 100 0001 to guarantee the automatic temperature control of the water bath during the entire sonication process Magnetic Ultrasound Emitter Maintenance e The ultrasound waves are created from a series of magnets that are attached to the water tank This system is very sensitive to the heat generated during a run e Do not exceed 2 hours of total sonication per run It is critical that the machine is allowed to cool at least 20 minutes between runs Damage resulting from non compliance to manual Instructions will void the warranty and shorten the lifespan of the machine e Ultrasound Emitters can be damaged by tilting or jarring the machine Exercise care If moving water tank Validated tubes for the Bioruptor NGS e DNA shearing Bioruptor NGS 0 65 ml Microtubes for DNA Shearing Cat No WA 005 0500 Fitting 0 65 ml tubes in the tube holder 1 Place the tubes on the co
8. 0 0001 ensures that water will only be replaced during the off cycle to avoid any interference the between water flow and the sonication process e Sonication time Minor adjustments in cycle number may be made to optimize results for various sample types and concentrations Cycle number listed above is a recommended guideline Actual results may vary depending on the amount and type of starting material concentration viscosity and or plastic tubes Diagenode recommends setting up a time dose response experiment for determining appropriate cycle number Larger length starting material e g total genomic DNA and higher concentration may require a longer dose to ensure a homogeneous shearing result e Water bath The sonication water bath is a critical component of the Bioruptor sonication system 1 Water purity Contaminants such as algae and particules may alter the ultrasonic waves propagation resulting in broader size distribution or larger peaks Bath water should be pure distilled water changed regularly 2 Water bath maintenance The water bath metal surface is fragile and requires a careful maintenance Use only soft sponge to remove traces Never use scratch scrub sponge since this would alter the ultrasonic wave emitter surface Supplementary Data Please note that there are three main sources of variation in both peak base pair size and distribution 1 The physical process of DNA fragmentation might not be entirely random in AT
9. 0Hz 2 1A EU 4 2A US Ultrasonic wave frequency 30 60 kHz Ultrasonic wave output power 25 210 W Transformer 220 100V dimensions Internal Control unit dimensions 350 W x 260 D x 165 H mm Sonication unit dimensions water bath 175 W x 160 D x 280 H mm Soundproof box dimensions 350 W x 350 D x 500 H mm Water bath volume 790 ml Timer Digital Possibility to control water flow via connector kit for water cooler Yes Tube holder Available for 0 55 ml tubes Total weight 30 kg Number of samples to be processed simultaneously 0 65 ml tubes 12 DIAGENODE BIORUPTOR NGS USER MANUAL PAGE 7 Getting to Know Your Bioruptor NGS System Bioruptor Components Overview Control Unit Water bath Motorized lid Soundproof box Power Cable EU Power Cable US Control Unit Cable Tube holder Bioruptor Water Cooler a To learn more please visit www diagenode com Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 8 DIAGENODE BIORUPTOR NGS USER MANUAL Connector kit for Water Cooler LS Solenoid valve with Plastic connector for Male nozzle connector Tubing connector cable motorized lid a a Open end wrench N 14 Open end wrench N 17 Plumbing Teflon Spare Tubing To learn more please visit www diagenode com Water bath The water bath is a critical component of the instru
10. ance amp Ordering Information Diagenode s a BELGIUM EUROPE Diagenode Inc USA NORTH AMERICA Avenue de Uhdpital 1 400 Morris Avenue Suite 101 Tour GIGA 3rd Floor Denville NJ 07834 USA 4000 Liege Belgium Tel 1 862 209 4680 Tel 32 4 364 20 50 Fax 1 862 209 4681 Fax 32 4 364 20 51 techsupport na diagenode com techsupport diagenode com orders naUMdiagenode com ordersOdiagenode com For a complete listing of Diagenode s international distributors visit http www diagenode com en support distributors php For the rest of the world please contact Diagenode s a DIAGENODE BIORUPTOR NGS USER MANUAL PAGE 3 Contents Critical Steps for Maintenance and Efficient Shearing kk kK eee ees 4 noida nee Hen beer a eee ee ee eee eae ee She ee ame ene eee 5 Bioruptor Technical Specifications lt 3 2c ence eccucsde ariete raras 6 Getting to Know Your Bioruptor NGS System sk kk kk kk eens 7 Bioruptor Components DVI W ora eG ale ied na SEY ec Brae aha K e ele a 7 Bloruptor Waler COOLER sesu i rra weeny a Bow de aE hte were Caress Se ye Rea eR ee es 7 Connector KI Tor Water COO S cirrosis l wal wl Wt Kl wd Ee oe ee eee dt eee eke ds 8 N gt N E EDMMMDMIAMMMMJ JMMIM M N Mu are HE NHNNN NIDjI jJMMiM k MMMMMMJJJ JM N 8 ND NN MMMMMMDMDM DM MJ MM gs ue Ge ere JM n a a NMn MI O _ QK MM MMNEa NENE MDM JM DP Y 9 Metal SOUND orto a Kawe n 0000 PA A a pad 9 Porn
11. are recommended guidelines and actual results may vary depending on the type and amount of starting material purity level concentration and or sample viscosity It is highly recommended that a time course response experiment be carried out e g varying the time of on and off durations as well as the number of cycles to determine the appropriate treatment for your specific sample Starting material with a smaller sample volume and a greater concentration than the recommended range may require a different time course to ensure homogenous shearing results Important comments about DNA shearing The Diagenode ACT Adaptative Cavitation Transfer technology process is highly reproducible However attention must be paid to the following treatment attributes to ensure best results e Tubes At present the recommended tube vessels are the Diagenode s Bioruptor NGS 0 65 ml Microtubes for DNA Shearing Cat No WA 005 0500 Pay attention not to damage the cap when closing the tubes since this could alter sonication results e Sample volume The recommended volume of the Diagenode s Bioruptor NGS 0 65 ml Microtubes for DNA Shearing Cat No WA 005 0500 is 100 ul When using lower volumes e g lt 50 ul less reproducible results may be observed due to an alteration of the ultrasonic waves distribution in the sample fluid thus reducing the efficiency of sonication which may result in broader size distribution or larger peaks e Sam
12. ater Cooler Cat No BioAcc Cool has to be used in combination with the Connector kit for Water Cooler Bioruptor Plus amp NGS Cat No VB 100 0001 to guarantee the automatic temperature control of the water bath during the entire sonication process Figure 2 The cooling system features two pumps and produces a regular water flow to maintain a constant water level in the tank The regulating valve Connector kit for Water Cooler ensures that water will only be replaced during the off cycle to avoid any interference between water flow and the sonication process ps A o Bioruptor Zs Connector kit for Water Cooler a O a Bioruptor NGS gt gt nm jili T n ll J Bioruptor Water Cooler Water bath Control Unit Fig 2 Setup of the Bioruptor NGS System including Bioruptor Water Cooler and Connector kit for Water cooler Note You may permanently install the Bioruptor in a cold room though this is not sufficient to avoid the temperature increase during sonication Motorized Lid The motorized lid along with the gear plate accessory keeps the sample tubes in constant rotation and ensures optimal position in the water bath during sonication When in motion do not hamper the rotation of the blue gear plate Avoid the immersion of the motor into the water Do not heat the blue plastic as it will warp Metallic Soundproof Box This metallic soundproof box absorbs very efficiently the noise generated
13. bit anti IgG rabbit rxns per kit 16 or 100 16 or 100 16 or 100 AB Auto01 A016 AB Auto02 A016 AB Auto03 A016 Cat N AB Auto01 G016 AB Auto02 G016 AB Auto03 G016 re AB Auto01 A100 AB Auto02 A100 AB Auto03 A100 AB Auto01 G100 1 Validated on SX 8G IP Star and SX 8G IP Star Compact Automated Systems 2 DNA purification has to be carried out with the IPure kit Cat No AL 100 0100 not included in the kit Diagenode Inc North America Phone 1 862 209 4680 Fax AB Auto02 G100 AB Auto03 6100 Ji 1 862 209 4681 Mail orders na diagenode com PAGE 20 DIAGENODE BIORUPTOR NGS USER MANUAL Manual ChIP kits Features Optimized for Suitable for ChIP seq and ChIP on chip Amount of cells IP Total Time of Assay Handling time Buffers and reagents for Control antibodies Control PCR primer pairs DNA purification rxns per kit Cat No LowCell ChIP kit Magnetic bead based protocol for all DNA protein interaction fast increased DNA yield All DNA protein interactions Yes 1 1 000 1 million Cell lysis chromatin shearing IP DNA purification anti IgG rabbit human TSH2B c fos myoglobin exon 2 DNA isolation buffer 16 or 48 kch maglow A16 kch maglow G16 kch maglow A48 kch maglow G48 Highcella ChiP kie pS teo RE kit Magnetic bead based protocol Ideal to recover large amount of DNA transcription factors ChIP on chip and to a
14. by the ultrasonic waterbath Tube Holders The 0 5 0 65 ml tube holder allows for simultaneous sonication of up to 12 DNA samples The optimal volume for DNA shearing is 100 ul and Bioruptor NGS 0 65 ml Microtubes for DNA Shearing Cat No WA 005 0500 are recommended for processing your samples PAGE 9 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 10 DIAGENODE BIORUPTOR NGS USER MANUAL Equipment Installation The following pages contain information on installing your particular Bioruptor NGS model This equipment must only be installed by personnel after reading this section Consider all hazards even though no particular hazards have been identified during installation Before starting installation work turn the main switch off beyond power connection and secure the unit against being re energized No special tools are required Three 3 square meters are needed to set up the Bioruptor Devices are designed to be safe under the following conditions Indoor use Altitude up to 2 000 meters Operating external temperature 0 C to 25 C Maximum relative humidity 80 Transient overvoltage typically present on the MAINS supply Degree of protection IP20 Installation overview 0 5 0 65 ml tube holder Motorized lid Metallic soundproof box Start button Level meter Digital timer Stop button Power button Water
15. echanical stress to shear DNA Ultrasound waves pass through the sample expanding and contracting the liquid During expansion negative pressures pull the molecules away from one another to forma cavity or bubble This process is called cavitation The bubble continues to absorb energy until it can no longer sustain itself and implodes This produces intense focused shearing forces that disperse and break biomolecules The fragmentation of DNA takes place as a consequence Transducer of this mechanical stress or shear Ultrasound Fig 1 Propagation in 0 65 ml tubes With the Bioruptor the entire volume of water present in the water bath is exposed to ultrasound allowing all the samples to be efficiently sonicated in parallel Figure 1 Use of Bioruptor by pregnant women Exposure to 30 60 kHz sound waves has not been shown to be harmful to human health However we would recommend avoiding unnecessary exposure Diagenode recommends that pregnant women should not be exposed to 30 60 kHz wave lengths for a long period of time Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 6 DIAGENODE BIORUPTOR NGS USER MANUAL Bioruptor Technical Specifications Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com Power Supply 100 230V 50 6
16. holder for 0 5 0 65 ml tubes Cat No UCD pack 0 5 and the corresponding Bioruptor NGS 0 65 ml Microtubes for DNA Shearing Cat No WA 005 0500 To use the tube holder remove the lower part by turning counterclockwise Then place microtubes in the unit Attach the lower part to the upper part of the adaptor To guarantee homogeneity of DNA shearing the tube holders should always be completely filled with tubes Never leave empty spaces in the tube holder Fill the empty spaces with tubes containing the same volume of distilled water 0 5 0 65 ml tube holder Cat No UCD pack 0 5 Bioruptor NGS 0 65 ml Microtubes for DNA Shearing Cat No WA 005 0500 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 16 DIAGENODE BIORUPTOR NGS USER MANUAL Standard protocols for DNA shearing 1230 bp 319 bp 1036 bp 234 bp 200 bp 100 150 200 500 600 1000 2000 Operating conditions Programmable DNA size distributions excellent reproducibility and high dsDNA yields with Bioruptor NGS Figure shows different DNA size distributions of sheared genomic DNA produced by varying the duration of sonication using power setting high H The different curves depict a specific Bioruptor NGS run optimized to produce specific mean sizes and size ranges for Next Generation sequencing All samples were analyzed on Bioanalyzer 2100 using DNA High Sensitivity
17. igh specificity monoclonal 5 meC Ab agarose beads fast qPCR linear amplification and genome wide analysis microarray sequencing Depending on the DNA purification method selected 1 pg 2 or 3 days 2h Methylated and unmethylated BAC clones Methylated DNA control unmethylated DNA control TSH2B GAPDH mc green 003 hMeDIP kit Quality control using internal controls improved handling magnetic beads high specificity monoclonal 5 hmC Ab fast qPCR linear amplification and genome wide analysis microarray sequencing Depending on the DNA purification method selected 1 pg 2 or 3 days Loh Hydroxymethylated methylated and unmethylated BAC clones Hydroxymethylated DNA control methylated DNA control unmethylated DNA control Sfil for genomic DNA 16 AF 104 0016 AF 110 0016 AF 111 0016 MagBisulfite kit Gives precise information on methylation status of single cytosines High conversion rate 399 DNA purification based on magnetic beads and compatible with SX 8G IP Star Automated System qPCR sequencing microarray l ng 1 yg 3 5 hours 2h Bisulfite specific primer pair 24 AF 106 0024 Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com CL 70 02 SLA SON 48 VIN Ordering information Description Cat No Bio
18. irm or ESC to escape without saving the change IMPORTANT NOTE Minimum time of the off cycle 30 seconds Digital Timer After the introductory message the control screen shows the main sonication parameters in the first two lines CYCLE Number Time and summary of actions in the last line see example below CYCLE Num 10 BIORUPTOR RUNNING ON Time ON 30sec wy 6 kk r kk OFF Time OFF 30sec CYCLE Num 04 10 re _ Press OK Modify gt gt TIME TIME ON 4 OFF 1 Cycle number reached A in thi CYCLE 4 cycles in this example START The display shows cycle 4 of 10 Bioruptor NGS will sonicate as shown Buttons and their functions Button A Pause restart after pausing Button B Press and hold this button during sonication to display T1 total on time per cycle and T2 total off time per cycle ESC Cancels previous selection OK Validate selection Decrease selected parameter move down Increase selected parameter move up Once all parameters are selected and confirmed press START Once the run is started BIORUPTOR RUNNING is displayed on the control screen Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR NGS USER MANUAL PAGE 15 Tube holders amp tubes For DNA shearing we highly recommend to use the tube
19. ite kit The Bioruptor with its powerful yet gentle ultrasound technology allows for consistent shearing a narrow size range of sheared DNA or chromatin and sample preservation necessary for successful experiments Antibodies Diagenode offers a large selection of optimized ChIP amp ChIP seg grade as well as MeDIP amp MeDIP seq grade antbodies that we have developed and characterized in house ji ME 1 For a complete listing of Diagenode s antibodies please visit www diagenode com for more information M 7 Chromatin Immunoprecipitation kits Automated ChIP kits Auto ChIP kit 1 Auto Histone ChIP seq kit 1 Auto Transcription ChIP kit 1 Optimized for working with histone Optimized for working with TF All DNA protein interaction saving Features ee antibodies in ChIP seg experiments anbosies saving time maximum time maximum reproducibility LE l PA aye saving time maximum reproducibility reproducibility Optimized for All DNA protein interactions Histones and histone modifications Transcription factors and co factors Downstream applications qPCR qPCR sequencing arrays 2 qPCR sequencing arrays 2 Amount of cells IP 1 000 1 million 1 000 10 million 1 000 10 million Total Time of Assay 1 day 1 day 1 day Handling time 30 min 30 min 30 min Buffers and reagents IP DNA Isolation IP IP Control antibodies DNA purification anti IgG rabbit DNA isolation buffer DIB anti IgG rab
20. l 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com Features Downstream applications Amount of DNA rxn Total Time of Assay Handling time Internal controls Control PCR primer pairs rxns per kit Cat No DIAGENODE BIORUPTOR NGS USER MANUAL Automated DNA methylation kits Auto MethylCap kit 1 Allows to capture fractions of methylated DNA by CpG density Includes control primer pairs for assessment of capture efficiency qPCR linear amplification and genome wide analysis such as microarray and sequencing 2 TSH2B GAPDH 48 AF Auto01 0048 Auto MeDIP kit 1 Quality control using internal controls improved handling magnetic beads high specificity monoclonal 5 meC Ab fast qPCR linear amplification and genome wide analysis such as microarray and sequencing 2 Methylated and unmethylated BAC clones Methylated DNA control unmethylated DNA control 16 or 100 AF Auto0 AF Auto0 AF Auto0 AF Auto0 1 A016 1 G016 1 A100 1 G100 1 Validated on SX 8G IP Star and SX 8G IP Star Compact Automated Systems 2 DNA purification has to be carried out with the IPure kit Cat No AL 100 0100 not included in the kit PAGE 21 Auto hMeDIP kit 1 Quality control using internal controls improved handling magnetic beads high specificity monoclonal 5 hmC Ab fast qPCR linear amplification and genome wide analysis such
21. ler Then press power button on the front side of the device Temperature can be set by pushing and turning the knob on the first side of the device Note See Bioruptor Water Cooler manual for additional information Now you are ready to start PAGE 13 Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 14 DIAGENODE BIORUPTOR NGS USER MANUAL Controlling the Sonication Start Button Begins sonication Intensity Level Meter Visualizes the power delivered by the control unit to the water bath relative scale Digital Timer Allows the user to easily program the sonication of samples ON OFF pulse time amp total time See use of digital timer in next section below Power Switch Turns Bioruptor on and off info diagenode com vi atonal na Stop Button Intensity Setting Button Cancels sonication Adjusts intensity of the generated ultrasonic waves Switch on power switch front side of control unit CYCLE NUMBER TIME ON TIME OFF and SONICATION INTENSITY are the parameters controlling the sonication First press or depending on the value to be modified The five flashing black squares move up or down Once you have selected the parameter to be modified press OK again The five flashing black squares disappear and 2 digits start flashing The digits can be changed by pressing or To select and save the correct number press OK to conf
22. ment The generators below the tank produce ultrasonic waves which are then transferred through water The water bath requires special handling and care as described below Handling The water bath must remain upright at all times especially when moved Tilting the water bath or handling roughly may damage the ultrasound emitter component resulting in a substantial drop in sonication efficiency If transportation of the apparatus Is required after initial set up it is Imperative to keep the tank at a right angle to the ground during the transport at all times Water level and quality The level of the water has been optimized and should always reach the red line sticker on the wall of the tank Distilled water should be used to fill the tank Replacement stickers can be obtained from Diagenode Water temperature The water in the water bath must be kept at 4 C Ultrasonic waves produced by the Bioruptor generate heat Drop off in sonication efficiency will occur above 10 C To ensure preservation of the samples and to prevent damage to the instrument it is necessary to start the sonication process with cold water and to keep it at 4 C during the sonication process Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR NGS USER MANUAL Automatic temperature control The Bioruptor W
23. nserted in the plastic connectors IMPORTANT NOTE If you need to cut a part of the tubing always use the provided Cutting Device Never use scissors because pieces of tubing with bad cuts generate leaks at junctions Only properly cut tubes can be inserted in the connectors The length determines the water level in the sonication bath After installation if the water level is not correct use the spare tubing and cut it at the right length The suction pipe red pipe must be set at the same level as the water level sticker is Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 12 DIAGENODE BIORUPTOR NGS USER MANUAL 6 Place the motorized lid a on the top of the water bath and connect it b Bioruptor Water Cooler amp Connector kit Location Requirements The Water Cooler must be located underneath the Bioruptor water bath Male nozzle connector Solenoid valve 7 Unpack the Water Cooler and remove the black protection rubber which connects Inlet and outlet 8 Place the solenoid valve and the male nozzle connector on the outlet and inlet as shown in the picture Use an open end wrench N 14 to install the male nozzle connector and N 17 to install the solenoid valve Seal each thread by adding Plumbing Teflon Note Red connectors and red tubing will ensure the waterflow from the water bath to the Water Cooler Blue connector and blue t
24. ple concentration Diagenode recommends using DNA concentration ranging between 1 and 20 ng pl 10 ng ul recommended Using larger concentration e g 50 100 ng l may result in broader peaks or variable peak distribution e Sample preparation Sample viscosity may have a major impact on sonication results Careful resuspension of DNA sample Is strongly recommended before sonication processing Multiple pipetting and gentle vortexing followed by a short centrifugation to recover sample volume at the bottom of the tube is therefore strongly recommended Storing DNA samples on ice during 10 15 minutes before sonication has also been shown to improve reproducibility e DNA quality DNA quality and quantity must be considered carefully since bad quality and quantity DNA may have several impacts on sonication and next gen sequencing downstream applications First DNA contamination e g from superfluous nucleic acids such as RNA small nucleic acid fragments excess proteins or other contaminating materials may interfere with DNA measurement method leading to incorrect DNA quantitation thus Also contaminating RNA in genomic DNA preparation might generate a biased fragment distribution profile on microfluidics based platform e g Agilent Bioanalyzer or alter sonication effiency Therefore it is highly recommended to use only high quality DNA when sonicating DNA for Next Gen sequencing Library preparation The DNA sample to be processed should be highly pu
25. re having an OD260 280 ratio of between 1 8 and 2 0 and should be as intact as possible DNA extracted using standard techniques e g Proteinase K digested double phenol chloform extraction ethanol precipitated treatment with RNase DNase free enzymatic digestion to remove contaminant RNA or commercial spin column based kits are recommended e Water temperature Propagation of ultrasound in a liquid unavoidably produces heat that can ultimately alter DNA sample e g by thermal denaturation To ensure the best preservation of the sample it is recommended to start the sonication process with cold water in the water bath During sonication especially when doing long sonication runs the temperature must also be controlled This is obtained by the automatic temperature control Diagenode Inc North America Phone 1 862 209 4680 Fax 1 862 209 4681 Mail orders na diagenode com PAGE 18 DIAGENODE BIORUPTOR NGS USER MANUAL Note The permanent installation of the Bioruptor in a cold room is possible although not sufficient to avoid the temperature increase due to sonication e Automatic temperature control A recirculating water cooler is used to guarantee the automatic temperature control of the water bath during the whole sonication process This water cooler Cat No BioAcc cool produces a regular water flow with a constant water level in the tank An additional regulating valve Single cycle valve for Water Cooler Cat No VB 10
26. rresponding tube holder 0 5 0 65 ml tube holder Cat No UCD pack 0 5 Never leave empty spaces in the tube holder Fill the empty spaces with tubes containing the same volume of water Screw the lid on the tube holder without over tightening the lid 2 Carefully place the tube holder on the holding plate 3 During sonication samples must remain at the bottom of the tube If needed briefly centrifuge samples during sonication after pausing the run Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR NGS USER MANUAL PAGE 5 Introduction Diagenode s Bioruptor NGS uses a gentle method of sonication to retain the integrity of DNA The Bioruptor Sonication System uses a water bath to generate indirect sonication waves which emanate from an ultrasound element below the water tank Because the system is gentler than other sonicators the Bioruptor produces better and more consistent results than with harsher sonication methods Up to 12 closed tubes can be sonicated in parallel and the continuous rotation of tubes allows even distribution of the energy for efficient sonication The Bioruptor enables automation of sonication guaranteeing higher reproducibility of results The effect of ultrasound on biological samples The Bioruptor sonication system uses ultrasound to create focused m
27. ruptor Bioruptor NGS with 0 5 0 65 ml tube holder UCD 600 TS Tube Holders 0 5 0 65 ml tube holder for Bioruptor Standard Bioruptor Plus amp Bioruptor NGS UCD pack 0 5 Cooling System Bioruptor Water Cooler including standard Bio cc cool connectors for Bioruptor Connector kit for Water Cooler Bioruptor Plus amp Bioruptor NGS VB 100 0001 Tubes a Bloruptor NGS 0 65 ml Microtubes for DNA WA 005 0500 Shearing Bioruptor Zs o siagenctle diagengte Innovating Epigenetic Solutions Diagenode s a BELGIUM EUROPE Avenue de l h pital 1 Vou GG sive Flecir 4000 Liege Belgium Tel 32 4 364 20 50 Ea 2 43640205 ordersediagenode com infoediagenode com Diagenode Inc USA NORTH AMERICA 400 Morris Avenue Suite 101 Denville NJ 07834 USA Tel 1 862 209 4680 Fax 1 862 209 4681 orders naediagenode com info naediagenode com For a complete listing of Diagenode s international distributors visit http www diagenode com pages distributors html For rest of the world please contact Diagenode s a e O 2010 Diagenode Inc All rights reserved The content of this document cannot be reproduced without prior permission of the authors Bioruptor and P Star are registered trademarks of Diagenode Inc Bioanalyzer is a registered trademark of Agilent
28. ubing will ensure the waterflow from the Water Cooler to the water bath 9 Connect the Bioruptor water bath to the Water Cooler with the red and blue tubes by inserting them in the appropriate connectors Optional Cut the length you need for the output and input flow Make sure there is enough slack The red and blue tubes must go through the soundproof box holes before being connected to the motorized lid see picture 10 Place the water bath into the soundproof box Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pital 1 4000 Li ge Sart Tilman Belgium Phone 32 4 364 20 50 Mail info diagenode com DIAGENODE BIORUPTOR NGS USER MANUAL 12 Plug the control unit cable into the control unit Make sure the red arrow on the plug Is pointing up 11 Place the control unit on the top of the soundproof 13 Plug the power cable into the control unit box 14 Plug the solenoid valve cable into the control unit 16 Fill the water bath up to the red fill line with distilled 15 Plug the power cable into the outlet and switch on the wares power switch on the back of the control unit 17 Plug the power cable into the back side of the Bioruptor Water Cooler Fill the tank of the circulator with 4 5 litres of water Distilled water should be used in order to minimize the proliferation of bacteria 18 Press first the power button on the back side of the Bioruptor Water Coo
29. void biais due to amplification steps Standard protocol with agarose beads optimized for transcription factors Transcription factors All DNA protein WANENA interactions Yes 1 Yes 1 10 million 1 million 1 day 3 days Loh 2h Cell lysis chromatin shearing IP DNA purification Cell lysis chromatin shearing IP anti IgG anti H3 K4me3 BMX c fos beta human TSH2B actin myoglobin GAPDH promoter exon 2 DNA isolation buffer Not included 16 18 kch mahigh A16 kch redTBP 012 kch mahigh G16 la Histone ChIP kit Standard protocol with agarose beads optimized for histones and their modifications Histones and histone modifications Yes 100 000 3 days 2h Cell lysis chromatin shearing IP anti TBP or RNA Pol Il 2 GAPDH 0 5 kb idem 1 kb c fos beta actin myoglobin exon 2 Not included 18 kch orgHIS 012 1 DNA purification has to be carried out with the IPure kit Cat No AL 100 0100 not included in the kit 2 Please mention your choice of antibody on your purchase order OneDay ChIP kit Protocol using agarose beads quick and ready to use on large quantities of sheared chromatin fast All DNA protein interactions Yes 1 5 2x 10e6 2h IP DNA purification anti IgG rabbit DNA purifying slurry 60 180 kch oneDIP 060 kch oneDIP 180 Europe Diagenode sa CHU Tour GIGA B34 3 tage Avenue de l H pita
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